Human chromodomain protein Y-like 2, Chromodomain

Target ID:

CDYL2

Deposition Date:

Wednesday 26th September 2012

Families:

Methyl-lysine readers

Related Diseases:

Chromatin and epigenetics

Structure type:

apo

Download Coordinates:

4HAE

Authors:

Qin S, Dombrovski L, Tempel W, Dong A, Xu C, Bountra C, Arrowsmith CH, Edwards AM, Min J, Wu H

Site:

Toronto

4HAE

tabs

Structure Details

In vertebrates, HP1-like chromodomains are present in the chromodomain Y
chromosome (CDY) family of proteins adjacent to a putative catalytic motif. The human
genome encodes three CDY family proteins, CDY, CDYL, and CDYL2. These proteins
have putative functions ranging from establishment of histone H4 acetylation during
spermiogenesis to regulation of transcription co-repressor complexes.
The CDY family of proteins contain two functional domains: a chromo domain involved
in chromatin binding and a catalytic domain found in many Coenzyme A (CoA)-
dependent acylation enzymes.

We have solved the crystal structure of human CDYL2 chromodomain at 2.0 Åresolution.

Materials and Methods

CDYL2

PDB:4HAE

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi229462825
Entry Clone Source:Open Biosystems
SGC Clone Accession:CDYL2:JMC01M-G09:C206444
Tag:N-terminal: His-tag with integrated TEV protease site:MHHHHHHSSGRENLYFQG
Host:E.coli BL21(DE3)-V2R-pRARE2

Construct

Prelude:
Sequence:MHHHHHHSSGRENLYFQGASGDLYEVERIVDKRKNKKGKWEYLIRWKGYGSTEDTWEPEHHLLHCEEFIDEFNGLHMSKDK
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:CDYL2 chromo domain was expressed inE.coli BL21(DE3)-V2R-pRARE2 in Terrific Broth (TB) in the presence of 50 µg/ml ofkanamycin and 30 mg/mL chloramphenicol at at 37°C to an OD600 of 2.0, the temperatureof the medium was lowered to 16 degree and the culture was induced with 0.4 mM IPTG.The cells were allowed to grow overnight before harvesting.

Purification

Procedure
The lysate was centrifuged at 16,000 rpm for 45 minutes and the supernatants weremixed with 8 mL 50% flurry of Ni-NTA beads and incubated at 4 degree on rotaryshaker for one hour. The mixture was then centrifuged at 2000 rpm for 5 min and thesupernatant discarded. The beads were then washed with 50 mL washing buffer, andfinally 25 mL the elution buffer. The protein further purified by a Superdex-75 gelfiltration column. Fractions containing the protein were collected and concentrated withAmicon Ultra-15 centrifugal filter. The purity of the preparation is tested by SDS-PAGEto be greater than 95%.

Extraction

Procedure
Cells were harvested by centrifugation at 6,000rpm. The cell pellets were frozen in liquid nitrogen and stored at -80°C.
= Extraction buffers =
20 mM Tris-HCl, pH 7.8, 250mM NaCl
= Extraction procedure =
Frozen cells from 2L TB culture were thawed and resuspended in 200 mL extractionbuffer and 8 uL benzonase (Sigma Catalog # E1014, 250U/uL), and lysed usingsonicator.
Concentration:50 mg/ml
Ligand
MassSpec:expected MW=9816.8 Da, not measured
Crystallization:Crystallization was setup in sitting drops with Red Wings screens initially. Diffractingcrystals were from initial screen plate for Red Wings F07.

Crystal used for structure determination was grown in 25% P3350 0.2M MgCl2, 0.1MTris buffer at pH 8.5.Crystals grow to a mountable size within 1 week
NMR Spectroscopy:
Data Collection:
Data Processing: